Project summary Bacteria have evolved complex strategies to compete and communicate with one another. One important mechanism of inter-bacterial competition is contact-dependent growth inhibition (CDI). CDI systems are found in a wide variety of Gram-negative bacteria, including many important human pathogens. CDI is mediated by the CdiB/CdiA family of two-partner secretion proteins. CdiB is an Omp85 outer-membrane protein that is required for the export and assembly of the CdiA exoprotein onto the cell surface. CdiA binds to receptors on susceptible bacteria and then delivers its C-terminal toxin domain (CdiA-CT) into the target cell. These systems also encode CdiI immunity proteins, which specifically bind to the CdiA-CT and neutralize toxin activity to protect CDI+ cells from auto-inhibition. CdiA-CT/CdiI sequences are highly variable, with >60 distinct toxin/immunity protein families recognized in bacterial genomes. We recently discovered that several CDI toxin/immunity proteins form higher order complexes with other cellular proteins. We hypothesize that these cellular protein function as permissive factors to activate CDI toxins inside target bacteria. The molecular mechanisms of CDI toxin activation are poorly understood, as are the broader physiological implications of toxin/permissive factor complexes. This application proposes a combination of genetic, biochemical and biophysical approaches to gain mechanistic insight into the network of protein-protein interactions that govern CDI. This research will significantly increase our understanding of the ecology and evolution of bacterial pathogens and could inform novel strategies for antimicrobial therapy.